Proteins are exceedingly versatile biomacromolecules. One class of proteins, known as enzymes, probably functions as nature's most effective catalysts for conducting complex organic syntheses. Because enzymes function so efficiently at low concentrations causing a high conversion of reactants to products under extremely mild conditions of temperature and pH and do so with an incomparable degree of specificity (in terms of functional groups affected), they have long been sought after as catalysts by organic chemists to conduct chemical reactions. Medicinal and pharmaceutical chemists have further been intrigued by the ability of enzymes often to synthesize just one of the possible optical isomers of a compound wherein the simple spatial arrangement of four different substituents on a particular carbon atom gives rise to unique pharmacological behavior. Interest in enzymes for synthetic purposes has also heightened recently by advances in molecular genetics that have allowed for the preparation of specific enzymes in a highly purified stated, on a relatively large scale, and at substantially reduced cost.
Insolubilization of enzymes without losing catalytic activity has been an objective of many investigators because of the very practical and obvious advantage that the catalyst system can be easily removed from the reaction mixture by simple filtration. Furthermore, although immobilization may seem "unnatural" for an enzyme, immobilized or heterogeneous conditions are frequently encountered by enzymes in their native state in vivo since enzymes are often physically located (literally immobilized) at interphases between aqueous and lipid regions within cell organelle structures.
Enzymes have traditionally been immobilized onto insoluble supports by three methods: 1) non-covalent adsorption by hydrophobic or ionic attraction to the support; 2) entrapment within a polymer matrix; and 3) by covalent attachment. Leaching or loss of the enzyme from the support is a serious problem and is often encountered with the adsorption and entrapping methods. Leaching results in physical losses of bound enzyme, normally the most expensive component of the system, and increased separation costs as well because the product solution is contaminated with free enzyme. Covalent attachment generally provides the lowest degree of enzyme leaching and often results as well in increased catalyst lifetime because of enhanced stabilization of the enzyme. Disadvantages of covalent methods include complexity of the chemical operations required to bind the enzyme and, more importantly, the relatively low degrees of catalytic efficiencies, e.g., 10-15%, often observed for bound enzymes compared to free enzymes.
Covalent attachment of enzymes to reactive supports has generally involved reaction of nucleophilic 4-aminobutyl groups of lysine residues within an enzyme's structure and electrophilic groups present on a support. A wide variety of electrophilic groups have been employed on supports for binding including azlactone, oxirane, cyanate, sulfonyl chloride, carbonyl imidazole, isothiocyanate, and isocyanate. Of the electrophilic groups listed, isocyanate is perhaps the most reactive and most desirable for protein binding, provided hydrolysis or reaction with the water solvent does not seriously compete with reaction by lysine residues. Hydrolysis is not only wasteful of isocyanate groups for binding but ultimately produces amine groups which become protonated and positively charged at useful binding pHs. Attraction of negative charges on the enzyme can result in temporary binding to this positively charged support via an ionic bond. However, if the pH or ionic strength of the medium is subsequently altered, ion exchange can occur and the enzyme will be released, i.e., leached, from the support.
An accepted tenet in the practice of binding enzymes to supports is that lipophilicity of the backbone support plays an important role in the overall activity and stability of the bound enzyme catalyst. Therefore, control and manipulation of support lipophilicity is important and may vary for a given enzyme. The simple procedure of changing the nature and relative amounts of hydrophilic and hydrophobic monomers in a free radical addition polymerization is especially conducive to efficient control of support lipophilicity. By contrast, lipophilicity control with step growth polymers such as polyurethanes and polyureas is not as easily achieved, especially when formation of support and protein binding from water are accomplished simultaneously.
Bozelli, et al., in U.S. Pat. No. 4,582,805 disclose thermoplastic, e.g., organic solvent soluble (uncrosslinked), homo- and copolymers of vinyl addition monomers containing isocyanate groups useful for immobilizing biological materials.